The development of new therapeutic agents for Hepatitis C virus (HCV) infection is a major public health priority. It is estimated that 170 million people worldwide, including approximately 4 million individuals in the United States, are infected with HCV. Available therapies are non-specific antiviral agents with modest efficacies and significant toxicities. We have selected HCV entry as a target for the discovery and development of novel antiviral agents. This stage of the virus life cycle represents an important target for drug development, and we have had success in the discovery and development of entry inhibitors for HIV that have now progressed to human clinical trials. In the Phase project, we generated novel HCV structural glycoprotein constructs that were used for the production of HCV pseudoparticles (HCVpp). We demonstrated that the HCVpp were able to specifically infect human liver cells, and that entry was sensitive to specific inhibitors. The HCV entry assay recapitulates the essential biology of this stage of the viral life cycle and may have potential utility for identifying new antiviral compounds. The overall goal of this Phase II project is to (1) adapt the entry assay to a high throughput screen (HTS) in 384-well format; (2) screen libraries of 100,000 diverse, drug-like compounds; (3) test active hits for breadth and potency of activity against genotypically diverse HCV isolates as well as a panel of unrelated viruses; and (4) determine the mechanism of action and molecular targets of inhibition in a panel of novel follow-on assays. Success in the Phase project is defined as the identification of 1 or more bona fide lead series of drug-like compounds that warrant optimization for potency, specificity and oral activity.